Bibliographic details of the publications referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined following the bibliography.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Genomic nucleotide sequences of HIV-1 strains referred to herein are represented by their corresponding DNA sequence.
Exemplary viral isolates referred to herein as "C18" and "C98" were deposited at the PHLS Centre for Applied Microbiology and Research, European Collection of Animal Cell Cultures (ECACC), Division of Biologies, Porton Down, Salisbury, Wiltshire SP4 OJG. C18 was deposited on Oct. 17, 1994 under Provisional Accession Number V94101706 and C98 was deposited on Oct. 31, 1994 under Provisional Accession Number V941031169. Viral isolate "C54" was deposited at ECACC on Mar. 10, 1995 under Provisional Accession Number V95031022.
A summary of particular deletion mutants of HIV-1 of the present invention referred to herein is given in FIG. 11.
Acquired Immune Deficiency Syndrome (AIDS) and AIDS related disorders are the clinical result of infection by Human Immunodeficiency Virus type 1 (Barre-Sinoussi et al, 1983). Infection by HIV-1 is generally characterised by progressive immune system damage (Teeuwsen et al, 1990; Clerici et al, 1989) leading to opportunistic infections, malignancies or wasting syndrome that constitute clinically-defined AIDS (Busch et at, 1991; Klaslow et al, 1990).
The high mortality rate of individuals infected with HIV-1 together with the social and economic consequences of the continuing HIV-1 epidemic has created an urgent need for a safe and effective treatment and/or prophylaxis against the devastating effects of AIDS. However, despite over a decade of high level scientific research into the pathogenesis of HIV-1 and the clinical manifestations of the disease, together with a detailed molecular analysis of the virus, there has been little success in the development of an effective vaccine. To date, the most effective therapy is treatment with zidovudine (AZT) which delays the onset of full blown AIDS and alleviates to some extent the symptoms of HIV-1 infection. However, AZT is not an innocuous compound and AZT, metabolic products thereof or impurities therein can cause a number of side effects which limit long term treatment with the drug. Furthermore, AZT resistant isolates have been reported during treatment. Clearly, therefore, a need exists to develop alternative strategies in preventing and treating HIV-1 infection.
The initial phases of HIV-1 infection are summarised by Levy (1993) as involving attachment, fussion and nucleocapsid entry. These phases have been the traditional foci in research into development of antiviral strategies. The molecular events at the virus genomic level have also been the subject of intense scientific research with an aim being the development of a live attenuated vaccine as a possible approach for the treatment or prophylaxis of HIV-1 infection.
There is a high variable rate of progression from initial HIV-1 infection to AIDS which reflects a rapidly changing pathogen and variable immune response of the host to infection (Sheppard et at, 1993). With regards to the latter, HIV-1 can be considered as a heterogenous group of viruses differing at the genetic level with concomitant variable pathogenicity. For example, HIV-1 strains can differ in their capacity to kill cells. Furthermore, it appears that HIV-1 strains evolve in a host after infection and that the evolution varies depending on the tissues infected by the virus. The major sites in the genome apparently responsible for biological and pathological variation are the highly variable envelope region (Cheng-Mayer et al, 1991; Shioda et al, 1992; Hwang, et al 1991; Sullivan et al, 1993; Groenink et al, 1993) and the viral regulatory regions such as tat (Leguern et al, 1993). The genetic complexity of the HIV-1 group of viruses together with their variable pathogenicity, are major difficulties in the development of live vaccines, genetic vaccines or component vaccines.
Notwithstanding the highly pathogenic nature of HIV-1, there are some reports of long term survival of subjects infected with the virus (Learmont et al, 1992; Levy, 1993; Sheppard et at, 1993; Lifson et al 1991). It is not always clear, however, whether a benign course following HIV-1 infection is due to host factors, viral factors or other unknown factors. There are reports that most infected people have at least laboratory evidence of progressive immune system damage in the form of CD4+ cell loss (Lang et al, 1989) and defective immune responses (Clerici et at, 1989).
Although simian monkeys have been used as an in vivo model for HIV and Simian Immunodeficiency Virus (SIV) infection, a moor handicap in AIDS research has been the absence of suitable in vivo models to study the pathogenesis of the disease and, in particular, to study the viruses involved in benign infection. The need for a suitable in vivo model is heightened by the fact that results obtained in vitro cannot necessarily be extrapolated to what occurs in vivo. This was clearly observed by Mosier et at (1993) where conflicting results were obtained in animals compared to cell cultures.
Despite the absence of suitable in vivo models, considerable scientific research has been directed to attenuating HIV-1 strains by mutagenesis of the virus genome. Deletions in the nef gene have been implicated in attenuated strains of SIV and their use in providing protective effects in monkeys (Daniel et al, 1992). However, there are conflicting reports on the possible negative influence the nef gene product has on the rate or extent of virus replication (Terwilliger et al, 1986; Luciw et al, 1987; Niederman et al, 1989; Kim et al, 1989; Hammes et al, 1989). In fact, Kim et al (1989) found that nef did not affect HIV-1 replication or HIV-1 long terminal repeat (LTR)-driven CAT expression. Kestler III et al (1991) found that the nef gene is required for full pathogenic potential in SiV. However, such is the complexity of the HIV-1 group of viruses and the variability of immune responses between individuals let alone different species that it is far from clear whether nef deleted strains of HIV-1 would behave similarly to nef deleted strains of SIV-I. There is a need, therefore, in order to investigate the possibility of nef deleted HIV-1 strain as a vaccine candidate, to identify individuals infected with such modified viruses.
Learmont et at (1992) reported that a cohort of five persons infected with blood products from a single HIV-1 infected donor have remained asymptomatic from up to about 10-14 years after infection. Subsequently, a sixth person has been identified as being part of the cohort. Both the donor and recipients were HIV-1 seropositive but with no indications of clinical symptoms of HIV-1 related disease and CD4+ cell number and .beta..sub.2 -microglobulin levels have remained in the normal range. The identification of this cohort of benignly infected individuals provides a unique in vivo model in which the pathogenesis of HIV-1 infection can be studied at the clinical and molecular biological levels.
However, it has not always been possible using conventional isolation procedures to routinely and reproducibly isolate viral strains from the above mentioned donor or recipients which has frustrated efforts to investigate the cause of the asymptomatic individuals. In accordance with the present invention, methods have now been established to isolate viruses from the above individuals. It has been determined, in accordance with the present invention, that individuals of the cohort are infected by non-pathogenic strains of HIV-1. Furthermore, the non-pathogenic strains of HIV-1 carry one or more nucleotide mutations. The non-pathogenic strains of the present invention enable the generation of a range of therapeutic, diagnostic and targeting agents against HIV-1 infection. The present invention also enables the attenuation of previously pathogenic strain of HIV-1. Additionally, an investigation of the immunological profiles of cohort individuals has revealed that a non-pathogenic strain of HIV-1 is indicated by a particular deletion in the coding region of a protein resulting in an altered immunological profile for the expressed protein. An example of an altered immunological profile results from a deletion of certain amino acids in the Nef protein.